The proposed research is a continued study of mobile group II introns and their applications in gene targeting. Mobile group II introns are catalytic RNAs ("ribozymes") that encode a reverse transcriptase and are thought to be evolutionary ancestors of spliceosomal introns, retroelements, and telomerase in higher organisms. They move to different genomic locations by using a novel site-specific DNA integration mechanism termed "retrohoming" in which the intron RNA reverse splices directly into a DNA strand and is then reverse transcribed by the intron-encoded protein. This mechanism is mediated by a ribonucleoprotein particle that contains the intron-encoded protein and the excised intron RNA and uses both for DNA target site recognition, with most of the specificity coming from base pairing of the intron RNA to the DNA target sequence. This feature combined with their high insertion frequency and specificity enabled us to develop mobile group II introns into highly efficient bacterial gene targeting vectors ("targetrons") with programmable DNA target specificity. As a result of work during the current grant period, targetrons are now widely used for gene targeting in bacteria, including medically and commercially important species in which gene targeting by other methods had been difficult or impossible. The proposed research will continue to combine basic and applied aspects. For the former, we will continue to study group II intron mobility mechanisms, focusing on the role of host factors, cellular interactions, and mechanisms by which group II introns proliferate within genomes. These studies will provide fundamental information about group II intron mobility mechanisms and their relationship to genome evolution, as well as underlie applications of group II introns in gene targeting. For the latter, we will build on progress during the current grant period to develop efficient group II intronbased gene targeting methods for higher organisms. We anticipate that such methods will have broad applications in genetic engineering, functional genomics, and ultimately gene therapy. Specific aims are: (1) To continue to study the role of host factors and cellular interactions in group II intron mobility. (2) To use group II introns that have multiplied within a thermophilic bacterium to investigate how group II introns proliferate within genomes and as a source of thermostable group II introns and RTs for structural studies and biotechnological applications. (3) To develop group II intron-based gene targeting methods for eukaryotic model organisms, focusing initially on Drosophila melanogaster. (4) To use Xenopus tropicalis to further develop methods for using group II introns for gene targeting in eukaryotes via sperm DNA modification. (5) To develop efficient group II intron-based gene targeting methods for mammalian cells. PUBLIC HEALTH RELEVANCE: The proposed research on genetic elements called mobile group II introns will provide novel insight into the function and evolution of the genomes of higher organisms and their viruses and could lead to the development of entirely new methods for genetic analysis and genetic engineering. These methods, which involve programmable site-specific modification of DNA, have potentially wide applications, including basic research into the etiology of human diseases, drug target-site validation, the production of therapeutic proteins, and gene therapy.